The work proposed in this application focuses on achieving a better understanding of the mechanism by which glucocorticoid receptors are transformed to the state that binds to nuclei. Glucocorticoid steroids bind to a cytosolic receptor to form a complex which then undergoes a temperature-dependent transformation that is thought to be necessary for subsequent nuclear events required for the hormone action. We have proposed that this transformation may result from dephosphorylation of the receptor protein or from dephosphorylation of some other, as yet identified, component of the binding complex. We are using both direct and indirect approaches to test the dephosphorylation model. The direct approach consists of attempting to label the receptor protein by incubating the intact cells with 32P, preparing cytosol, incubating the cytosol at 0 degrees C with (3H)triamcinolone acetonide, prepurifying the untransformed receptor by chromotography on DEAE-cellulose in the presence of 10 mM molybdate, and resolving phosphorylated protein by polyacrylamide gel electrophoresis and autoradiography. Both untransformed receptor and receptor that has been transformed by various methods will be analyzed in this way to see if phosphorylated and dephosphorylated forms can be visualized. The indirect approaches include: 1) attempting to transform the receptor by adding a purified phosphatase to cytosol; 2) determining whether an endogenous heat-stable inhibitor of transformation identified by others is acting as an inhibitor of phosphatase action.